www.nature.com/scientificreports OPEN Structural Basis for the Enhanced Anti-Diabetic Efcacy of Lobeglitazone on PPARγ Received: 9 October 2017 Jun Young Jang 1, Hwan Bae2, Yong Jae Lee3, Young Il Choi3, Hyun-Jung Kim4, Accepted: 4 December 2017 Seung Bum Park 2, Se Won Suh2, Sang Wan Kim 5 & Byung Woo Han 1 Published: xx xx xxxx Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily. It functions as a ligand-activated transcription factor and plays important roles in the regulation of adipocyte diferentiation, insulin resistance, and infammation. Here, we report the crystal structures of PPARγ in complex with lobeglitazone, a novel PPARγ agonist, and with rosiglitazone for comparison. The thiazolidinedione (TZD) moiety of lobeglitazone occupies the canonical ligand-binding pocket near the activation function-2 (AF-2) helix (i.e., helix H12) in ligand-binding domain as the TZD moiety of rosiglitazone does. However, the elongated p-methoxyphenol moiety of lobeglitazone interacts with the hydrophobic pocket near the alternate binding site of PPARγ. The extended interaction of lobeglitazone with the hydrophobic pocket enhances its binding afnity and could afect the cyclin- dependent kinase 5 (Cdk5)-mediated phosphorylation of PPARγ at Ser245 (in PPARγ1 numbering; Ser273 in PPARγ2 numbering). Lobeglitazone inhibited the phosphorylation of PPARγ at Ser245 in a dose-dependent manner and exhibited a better inhibitory efect on Ser245 phosphorylation than rosiglitazone did. Our study provides new structural insights into the PPARγ regulation by TZD drugs and could be useful for the discovery of new PPARγ ligands as an anti-diabetic drug, minimizing known side efects. Peroxisome proliferator-activated receptors (PPARs) are transcription factors activated by a group of ligands belonging to the thyroid/retinoid family (class II) of nuclear receptors1. PPARs regulate the transcription of target genes through heterodimerization with retinoid X receptors (RXRs) and binding to cognate peroxisome pro- liferator response elements (PPREs)2. PPARs, known as “lipid-sensing” receptors, are present in three subtypes (PPARα, PPARγ, and PPARδ/β) and regulate lipid and glucose homeostasis3. PPARγ is highly expressed in adi- pocytes and is also expressed in epithelial cells and macrophages. It plays important roles in adipocyte diferenti- ation, insulin resistance, and atherosclerosis4. PPARγ has a remarkably larger ligand-binding pocket (LBP) than other nuclear receptors5 and is known to be activated by a wide range of endogenous ligands and synthetic ligands. Although the physiological ligand of PPARγ has not been clearly elucidated yet, the endogenous PPARγ ligands reported so far include polyun- saturated fatty acids6, oxidized fatty acids7, nitrated fatty acids8, lysophospholipids9, and prostanoids such as 15-deoxy-Δ12,14-prostaglandin J210. Synthetic ligands of PPARγ can be classifed as full agonists, partial ago- nists, and antagonists. When full agonists bind to PPARγ, the activation function-2 (AF-2) helix (i.e., helix H12) changes its conformation and PPARγ recruits coactivator to induce adipogenesis and insulin sensitization11,12. PPARγ is also activated by partial agonists. However, partial agonists stabilize PPARγ ligand-binding domain (LBD) in a distinct manner compared with full agonists. Partial agonists stabilize the β-sheet region (β1, β2, and β4, and helix H3 preferentially, but leave AF-2 helix in a very dynamic state13. Antagonists exhibit high afnity but do not activate PPARγ, suggesting that the conformational change in AF-2 helix is not sufcient to allow coacti- vator binding or AF-2 helix exists in its inactive form14. 1Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, 08826, Republic of Korea. 2Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul, 08826, Republic of Korea. 3CKD Research Institute, Chong Kun Dang Pharmaceutical Corporation, Yongin, 16995, Republic of Korea. 4College of Pharmacy, Chung-Ang University, Seoul, 06974, Republic of Korea. 5College of Medicine, Seoul National University, Seoul, 03080, Republic of Korea. Correspondence and requests for materials should be addressed to S.W.K. (email: [email protected]) or B.W.H. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:31 | DOI:10.1038/s41598-017-18274-1 1 www.nature.com/scientificreports/ Figure 1. Chemical structures of lobeglitazone, rosiglitazone, and pioglitazone. PPARγ is a good therapeutic target for type 2 diabetes mellitus. When the thiazolidinedione (TZD) class of anti-diabetic drugs was frst reported in 1980s15, the molecular basis of TZD drugs was unclear. In 1990s, TZD drugs were found to bind to PPARγ16, and as mentioned above, various synthetic ligands for PPARγ have been developed so far. Full agonists such as TZD drugs are efective in treating type 2 diabetes mellitus, but adverse efects have been problematic, including fuid retention, edema, bone loss, and weight gain17. Te mechanism underlying the anti-diabetic efects of TZD drugs and their side efects has not been well understood. Recently, Choi et al. reported that the phosphorylation of PPARγ at Ser245 (in PPARγ1 numbering; Ser273 in PPARγ2 numbering) by cyclin-dependent kinase 5 (Cdk5) neither activates nor inhibits general transcriptional activity of PPARγ, but it changes the expression of specifc genes such as adiponectin and adipsin that are linked to insu- lin sensitivity18. TZD drugs have been known to strongly regulate both the Cdk5-mediated phosphorylation of PPARγ and the expression of PPARγ target genes18. Recent study has also revealed that the alternate binding site extending from the third arm of the known LBP of PPARγ could afect the phosphorylation of PPARγ at Ser24519. Tus, many researchers are trying to develop synthetic ligands that selectively inhibit the Cdk5-mediated phos- phorylation of PPARγ without classical transcriptional agonism20,21. Lobeglitazone (CKD-501; Chong Kun Dang Pharmaceutical Corp., Seoul, Republic of Korea) is an anti-diabetic drug of the TZD class known as a potent PPARγ agonist, and has been used in the treatment of type 2 diabetes mellitus22. Lobeglitazone is structurally similar to two well-known TZD drugs, rosiglitazone and pioglitazone, and was synthesized from the rosiglitazone backbone by substitution of the pyrimidine moiety for the pyridine part of rosiglitazone followed by addition of the p-methoxyphenol functional group at 4-position of the pyrimidine moiety (Fig. 1). Lobeglitazone showed better biological activities than known reference compounds (rosiglitazone and pioglitazone). Compared with rosiglitazone, lobeglitazone showed 100 times increased efcacy in the triglyceride accumulation experiments in 3T3-L1 cells and 2.4-fold and over 8-fold increased efcacies in the glucose and triglyceride lowering experiments in KKAy mice, respectively23. To our surprise, rosiglitazone and pioglitazone exhibit diferent clinical adverse events, which implies that slight changes in receptor-ligand interactions can cause signifcant pharmacological diferences in TZD drugs13. Tus, studies of PPARγ-ligand complex structures are cru- cial to better understand the mechanism of PPARγ modulation and it is also true with a more potent anti-diabetic drug, lobeglitazone. To elucidate a structural basis for understanding activities of lobeglitazone, we determined the crystal structure of PPARγ LBD in complex with lobeglitazone. For exact comparison of binding modes of lobeglitazone and rosiglitazone, we also determined the crystal structure of PPARγ LBD in complex with rosigl- itazone. Te overall binding mode of lobeglitazone to PPARγ LBD is very similar to that of rosiglitazone except the elongated p-methoxyphenol group of lobeglitazone. Notably, the elongated p-methoxyphenol group occupies the hydrophobic pocket near the alternate binding site of PPARγ. Lobeglitazone also blocked the Cdk5-mediated phosphorylation of PPARγ at Ser245 more potently than rosiglitazone did in vitro. Our results enhance the current understanding of PPARγ regulation by TZD drugs more in detail. Results Overall structure of lobeglitazone-bound PPARγ LBD and structural comparisons. To gain fur- ther insight on the binding mode of lobeglitazone, we solved the crystal structure of PPARγ LBD in complex with lobeglitazone in the presence of a peptide derived from the human steroid receptor coactivator-1 (SRC-1) coactivator protein at 2.15 Å resolution, using X-ray crystallography. For a comparative study, we have also deter- mined the rosiglitazone-bound structure of PPARγ LBD in the presence of the SRC-1 coactivator peptide. Te SCIENTIFIC REPORTS | (2018) 8:31 | DOI:10.1038/s41598-017-18274-1 2 www.nature.com/scientificreports/ Figure 2. Overall structures of PPARγ LBD in complex with lobeglitazone and with rosiglitazone. Lobeglitazone-bound and rosiglitazone-bound PPARγ LBD structures are superimposed and colored in salmon and yellow orange, respectively, all of which include the SRC-1 coactivator peptide colored same with bound PPARγ LBD structures. Lobeglitazone and rosiglitazone are represented as a stick model in green and pale cyan, respectively. Ser245 (PPARγ1 numbering; Ser273 in PPARγ2 numbering) is the Cdk5-mediated phosphorylation site, shown by red sticks. Regions with RMSD 2.0 Å and bigger are indicated with blue dashed circles. Overall structures are shown in stereo view. structures adopt the canonical fold of general nuclear receptors1,